Breast cancer is the second deadliest cancer in women, and will claim more than 40,000 lives in the United States in 2015 alone. Mammography is the first line imaging technique for early breast cancer detection, but the presence of dense breast tissue decreases its diagnostic accuracy. Due to its widespread availability and increased cancer detection rates, ultrasound is often performed as a second line test in women with dense breast tissue. However, ultrasound results in many false positive findings with unnecessary biopsies with increased associated health care costs. The goal of this research proposal is to develop an imaging approach that allows accurate, non-invasive characterization of focal breast lesions into clinically actionable (follow-up with biopsy or surgery is needed) and non-actionable lesions. We propose using spectroscopic photoacoustic (sPA) molecular imaging combined with a new clinically-translatable contrast agent that can be incorporated into the current clinical ultrasound imaging workflow. The new contrast agent will be developed by combining two clinically used components: First, a human fibronectin-based binding ligand (FN3-scaffolds), similar versions of which have been shown to be safe in patients; and second, the near-infrared fluorescent dye, indocyanine green (ICG), which is FDA approved for IV injection in patients. The ICG-FN3-scaffold will be targeted at a recently identified and validated breast cancer marker, B7-H3, which we have shown to be highly expressed in various types of human breast cancer compared to normal breast tissue and benign breast lesions in patients. It will be synthesized, tested, and optimized for its B7-H3 binding abilities in murine and human cell lines using flow cytometry. Since ICG can undergo significant shifts in optical absorption spectra based on its bound state to proteins, through live cell imaging experiments with confocal microscopy, we will assess whether B7-H3 receptor mediated endocytosis of the contrast agent results in intracellular release of ICG with consecutive spectral shift of its absorption spectrum. This shift could be leveraged to differentiate imaging signal from bound and non- bound contrast agent, thereby increasing tumor to background signal. Finally, to test the ability of B7-H3-targeted sPA molecular imaging to differentiate between clinically actionable and non-actionable lesions, a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer development will be used. Animals at varying ages, corresponding to different histopathological disease stages (normal, hyperplasia, DCIS, and breast cancer), will be imaged with sPA following IV injection of B7-H3-targeted ICG-FN3 and a sPA imaging signal threshold will be determined to allow differentiation of benign vs malignant lesions. This will be followed by a prospective study to assess the diagnostic accuracy of B7-H3-targeted sPA to differentiate clinically actionable from non-actionable lesions based on the sPA imaging signal in this model. Our study will lay the foundation for a significant change to current clinical breast imaging practice by improving non-invasive characterization of focal breast lesions, with the potential of substantially changing future management of breast cancer patients.